DESCRIPTION: The triplet state of tryptophan residues in proteins decays on the msec-sec time scale and is highly sensitive to the microenvironment of this residue. Long-lived room temperature phosphorescence (RTP) is invariably observed from tryptophan residues buried in the rigid, hydrophobic cores of globular proteins and can therefore be used to specifically probe aspects of these domains, including dynamics of interactions that occur on the time scale of the excited triplet state or longer. In his previous work, several triplet- state-based methodologies have been developed, demonstrated, and utilized to determine distances on the molecular level, to detect folding and unfolding protein intermediates with increased resolution, to observe heterogeneity of proteins in solution and to follow the slow annealing of a protein core domain during refolding. Dr. Gafni has strong indications from his work, as well as that of others, that this annealing may be a general phenomenon in proteins and is likely to cause the conformational modifications observed in proteins in cells of old animals. The long-term objective of this proposal is to apply triplet state-based methodologies to problems of structure, stability, and slow dynamics of proteins (such as the conformational changes that occur during folding or annealing of the protein core). The major research effort to be pursued in this proposed work will focus on the folding and stabilization of E. coli alkaline phosphatase in vitro as well as in vivo. The in vivo work is based on the exceptionally long-lived triplet state of this enzyme which enables its resolution from background luminescence from the cell. The research effort will place special emphasis on the role of individual amino acid residues in stabilizing the folded state and on the protein annealing mentioned above. To this end, he will prepare a number of AP mutants in which amino acid residues in the immediate environment of the phosphorescent tryptophan will be replaced by smaller residues. The structures of the mutants will be determined by x-ray crystallography, and the size of cavities formed by each mutation will be determined and correlated with protein stability, with several RTP parameters (lifetime, width of lifetime distribution, rate of D/H exchange) and with the rate of protein annealing. The latter variables are expected to trace the increased lability of the core due to cavity formation, as does the stability. The second research direction will focus on the relationship between changes in RTP decay and changes in structural details of proteins. In most cases, these changes are not observable by other means (e.g., UV-CD). Hence, Dr. Gafni proposes to determine the electronic energy transfer rate from the Trp triplet state via the exchange mechanism and measured by sensitized luminescence from Tb3+ bound to the metal binding sites of AP. This approach, which depends exponentially on the distance between Trp and Tb3+, has been demonstrated by Dr. Gafni and has potentially greater sensitivity over other methodologies.